Knowledge of the elastic properties of actin filaments is crucial for considering its role in muscle contraction, cellular motile events, and formation of cell shape. The stiffness of actin filaments in the directions of stretching and bending has been determined. In this study, we have directly determined the torsional rigidity and breaking force of single actin filaments by measuring the rotational Brownian motion and tensile strength using optical tweezers and microneedles, respectively. Rotational angular f luctuations of filaments supplied the torsional rigidity as (8.0 ؎ 1.2) ؋ 10 ؊26 Nm 2 . This value is similar to that deduced from the longitudinal rigidity, assuming the actin filament to be a homogeneous rod. The breaking force of the actin-actin bond was measured while twisting a filament through various angles using microneedles. The breaking force decreased greatly under twist, e.g., from 600-320 pN when filaments were turned through 90؇, independent of the rotational direction. Our results indicate that an actin filament exhibits comparable f lexibility in the rotational and longitudinal directions, but breaks more easily under torsional load.Actin is a major protein involved in a variety of cellular motile events and in the maintenance of cell shape and form. Determining its elastic properties in the polymeric state is central to an understanding of its function (1, 2). Recently, actin filaments have been measured to be several-fold more flexible longitudinally in vitro (3) and in muscle (4-6) than many models of muscle contraction have assumed (7). This finding would seem to require reconsideration of aspects of such models (8-10). Because of its helical structure, an actin filament should experience not only longitudinal but also torsional loads during interactions with myosin (11). To explain the mechanism of force generation, it is also important to know the elastic behavior of actin filaments during torsion. The torsional rigidity of actin filament can be estimated based on its bending rigidity, assuming the actin filament to be a homogenous rod (12). However, this assumption is not necessarily correct, because the actin filament is a double-helical polymer of globular actin monomers (13,14). Spectroscopic (12,(15)(16)(17) and electron microscopic studies (18) have suggested that the elastic property of an actin filament is largely anisotropic in the directions of twisting, bending, and stretching, i.e., the torsional rigidity is much smaller than the bending and longitudinal ones, assuming the actin filament to be a homogeneous rod, whereas the normal mode analysis based on the atomic structure of actin has shown much larger torsional rigidity than those suggested by these studies (19).Here, we have directly determined the torsional rigidity of actin filaments and the actin-actin bond breaking force under torsion by manipulating single actin filaments with optical tweezers and microneedles, respectively. Developments in video-assist fluorescence microscopy have enabled direct observat...